The overall goal of this application is to determine the importance of the cellular protein apolipoprotein E (apoE) in hepatitis C virus (HCV) infection and production in vivo and to define the underlying molecular mechanism of apoE in HCV infection and assembly. HCV chronically infects approximately 170 million people worldwide. HCV infection is the most common indication for liver transplantation in the United States. HCV is also the major cause of hepatocellular carcinoma (HCC), the most rapidly increasing cancer with more than 14,000 deaths each year in the U.S. Pegylated interferon-a in combination with ribavirin is the only option for treatment of hepatitis C. However, more than 50% patients infected with HCV genotype 1, the dominant virus accounting for ~70% infections, do not respond to IFN and ribavirin therapy. Therefore, there is an urgent need to develop more efficacious anti-HCV drugs. Identification of novel targets is key to discovery of new classes of antiviral drugs. We have demonstrated that apoE is a structural component of HCV and plays important roles in HCV infection and virion assembly. We have also demonstrated that apoE interacts with HCV NS5A and that the apoE-NS5A interaction is important for HCV assembly. More importantly, our preliminary studies found that apoE but not HCV E2 mediates HCV attachment. However, these findings were derived from the studies with a cell culture grown HCV of genotype 2a (JFH1). The significance of apoE in the HCV life cycle in vivo has not been experimentally examined. In specific aim 1, we will determine the importance of apoE in HCV infection and production in vivo. The HCV-neutralizing activity of an apoE-specific monoclonal antibody and the therapeutic efficacy of an apoE-silencing shRNA will be evaluated in a humanized HCV mouse model using clinical HCV isolates of genotype 1. In specific aim 2, we will define the underlying molecular mechanism of apoE in HCV infection. Specific domain and critical residues of apoE important for receptor-binding will be determined by mutagenesis studies. Additionally, we will identify specific cell surface receptor(s) that mediate apoE-binding and HCV attachment. In specific Aim 3, we will illustrate the mechanism of action of apoE in HCV assembly. Specifically, we will determine the molecular basis underlying the apoE-NS5A interaction and identify cellular mechanism and/or pathways responsible for the apoE-mediated HCV assembly. These specific aims will be successfully accomplished using multidisciplinary approaches, including a robust HCV reverse genetics system, site-directed mutagenesis, RNA silencing, cell biological, immunological, and biochemical methodologies. That HCV uses a cellular protein (apoE) for cell attachment is unprecedented and challenges the current dogma that viral envelope proteins mediate virus attachment by binding to cell surface receptors. New knowledge originating from these studies will result in a paradigm change regarding the roles of viral and cellular proteins in virus infection. The studies described in this application will also provide novel targets for anti-HCV drug discovery.